Well Plate Reactor

ABSTRACT

A well plate and its supporting devices provide capabilities found in larger fermenters, such as controlling the oxygen level, the pH level, and temperature of the contents of the well. The well plate includes a plurality of wells, each of which can be independently controlled. Apertures in the wells, for example, provide access for a gas supply and sensors within each well provide data relating to, e.g., oxygen and/or pH level in the well. A control system controls the gas supply for each well based on the information provided by the sensor within the well. Similarly, temperature control elements, such as a heater or cooler, is placed in thermal contact with the interior of the well, as is a temperature measurement element. A control system can independently control the temperature of the contents of the well based on information provided by the temperature measurement element for that well.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.12/024,973, filed Feb. 1, 2008, entitled “Well Plate Reactor”, which isa divisional application of U.S. patent application Ser. No. 10/777,581,filed Feb. 11, 2004, entitled “Well Plate Reactor,” now U.S. Pat. No.7,374,725, both of which are incorporated herein by reference in theirentireties.

FIELD OF THE INVENTION

The present invention relates to reactors, e.g., for cell culture,fermentation, and cell based assays and in particular to well plates andsupporting devices.

BACKGROUND

Cell culture and fermentation have value for many aspects of industrialproduction, such as pharmaceuticals, industrial enzyme production (e.g.detergents, food additives, textile processing, pulp and paperprocessing, grain processing incl. production of high fructose cornsyrup), potable and fuel ethanol, amino acids, vitamins, feed additives,and many others. The actual organisms in the fermenter may vary greatlyand can include a variety of bacteria, yeast, fungi, insect cells,mammalian cells, and others.

Conventionally, complex large-scale fermentation (hundreds of thousandsof liters) systems are used for production. Large scale systems aremanufactured by companies, such as Applikon, B. Braun, and New BrunswickScientific. Typically, large scale cell culture and fermentation systemsmust be capable of: 1) feeding the media with nutrients, 2) measuringand changing the Oxygen level, 3) measuring and changing thetemperature, 4) measuring and changing the pH level, 4) stifling thecontents, 5) purging byproducts (such as CO₂), and 6) monitoring thereaction quality (such as cell density and protein expression).

Before scaling up reactions in large capacity fermenters, similarreactions are typically performed at a smaller scale. Small scalefermenters, e.g., in the 1-20 liter range, provide most if not all ofthe desired performance functions of the large scale fermentersdescribed above. However, the small scale fermenters are expensive, andhave a relatively larger form than necessary for many desiredapplications.

For fermentations on a smaller scale, less expensive systems aretypically used. However, conventional inexpensive systems used for verysmall scale fermentation typically lose several of the desiredperformance capabilities and, accordingly, quality.

The two most common systems for smaller scale experiments are shakeflasks and micro-well plates. Shake flasks are simply glass or plasticvessels that are shaken and supplied with gasses to support the cellgrowth.

Micro-well plates (which are also called micro-titer plates, well platesor micro plates and will be referred to herein as “well plates”) aresimply molded plastic plates, with a plurality of wells. A separatefermentation can be performed in each well of a well plate. Well platestypically have a 96 well format, however other well plate sizes alsoexist (such as 24 well, 48 well, 192 well, 384 well, and 1536 wells).The shape and size of well plates are standardized. The standardizationis run by the Society for Bimolecular Screening (SBS).

The main drawback of well plates is that they are typicallyuncontrolled. While it is possible to run reactions and perform someoptical measurements in a conventional well plate, conventional systemsdo not allow for well-by-well control of conditions in individual wells.Further, many of the desired performance capabilities found in thelarger scale fermenters cannot be found in well plates, which inhibitsexperiments of the quality that are performed in larger fermenters.

By way of example, applications which would be desirable for well platesare drug discovery and diagnostic testing in which cell-based assays areused. Cell-based assays refer to any number of different experimentsbased on the use of live cells, such as measuring cell proliferation ormortality. There is a recent trend toward more cell-based assays in drugdiscovery since they are more reliable and robust than biochemicalassays. An example of this type of application would be screeningcompounds for use in cancer therapy. In this case, a particular cancercell line would be grown under controlled conditions. The growth rate ofthe cells would be measured after the introduction of a small quantityof test compound. Compounds that kill, or slow or halt growth versus acontrol are drug candidates. The same approach is used in toxicologyscreening to assess the potential impact of a compound on differenthuman tissues.

Unfortunately, many cell-based assays are difficult to perform inconventional well plates. The cell lines involved can be quite sensitiveto small changes in their environment, resulting in noisy assay output.Other desirable applications, such as diagnostic and clinical tests arelikewise difficult to perform in conventional well plates.

Accordingly, what is needed is an improved well plate design andsupporting devices that provides, e.g., the performance capabilities ofthe larger scale fermenters while remaining relatively low cost.

SUMMARY

In accordance with the present invention, a well plate and itssupporting instrumentation is used to provide the capabilities typicallyfound in larger fermenters, such as controlling the oxygen level, the pHlevel, and temperature of the contents of the well. The well plateincludes a plurality of wells, each of which can be independentlycontrolled.

In one embodiment of the present invention, a well plate includes aplurality of wells, each well being defined by at least one surface thatdefines a cavity having an opening. Each well includes at least oneaperture through a surface of the well, the aperture being configured toprovide a gas supply access to the interior of the well and at least oneof a pH level sensor and a dissolved oxygen sensor disposed within thewell.

In another aspect of the present invention, a well plate includes aplurality of wells, each well having at least one surface that definesan opening at a top of the well. Each well includes a first aperturethrough a surface, the first aperture being configured to provide a gassupply access to the interior of the well. Each well includes at leastone additional aperture through a surface, the at least one additionalaperture being configured to place one of a temperature control elementand a temperature measurement element in thermal contact with theinterior of the well.

In another aspect of the present invention, an apparatus, forcontrolling at least one of the pH level and dissolved oxygen in thecontents in a plurality of wells in a well plate, each well beingdefined by at least one surface that defines an opening and has anaperture, includes a gas supply for providing gas to a well through theaperture in the well. The apparatus also includes at least one detectorfor detecting the pH level and/or the dissolved oxygen in the contentsof a well and a control system that is coupled to the gas supply and thedetector. The control system controls the amount of gas supplied to thewell by the gas supply in response to the detected pH level and/ordissolved oxygen.

In another aspect of the present invention, an apparatus that is usedwith a well plate having a plurality of well, each well being defined byat least one surface that defines an interior cavity having an openingincludes a plurality of drip valves. There is at least one drip valveassociated with each well positioned over the opening of each well. Thedrip valves are configured to provide a liquid to the interior cavitiesof the associated wells. The apparatus further includes a plurality ofdetectors for detecting a property of the contents of the wells, whereinthere is at least one detector associated with each well and a controlsystem coupled to the plurality of detectors and the plurality of dripvalves. The control system controls the amount of the liquid provided bythe drip valves to the associated wells in response to the property ofthe contents in the associated wells detected by the detectorsassociated with each well.

In another aspect of the present invention, a method includes providinga well plate with a plurality of wells with content in each well. Themethod includes measuring the pH level and/or the dissolved oxygen inthe contents of each well and providing at least one gas to the contentsat least one well through a membrane and an aperture in the well inresponse to the measured pH level and/or dissolved oxygen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 illustrate a top perspective view and a top plan view of awell plate, in accordance with one embodiment of the present invention.

FIGS. 3A and 3B are views of the interior bottom surface of differentembodiments of a well.

FIG. 4 illustrates a perspective view of the bottom of the well platewith a membrane attached to the exterior bottom surfaces of the wells.

FIG. 5A illustrates a side view of two wells with a laminate membranestructure.

FIG. 5B illustrates a side view of two wells with membranes attached tothe interior bottom surface of the wells.

FIG. 6 shows a block diagram of a device that may be used with wellplate to control the culture and/or fermentation of the contents of thewells.

FIG. 7 illustrates a side view of one embodiment of vacuum clamping thewell plate.

FIGS. 8 and 9 illustrate a perspective view and side view, respectively,of a well plate mounted on an instrumentation block.

FIG. 10 illustrates a top perspective view of a support plate used inthe instrumentation block.

FIG. 11 is a top plan view of the optics plate used in theinstrumentation block.

FIG. 12 illustrates a side view of a well with a sensor and a detectionhead.

FIGS. 13A and 13B illustrate embodiments of the detection head.

FIG. 14 illustrates a top plan view of a detection head that usesoptical fibers and multiplexers.

FIG. 15 illustrates an embodiment of a detection head that uses a twodimensional stage.

FIG. 16 schematically illustrates a gas feedback loop for one well.

FIG. 17 illustrates top view of a portion of the well plate with a wellover the support plate and a temperature feedback loop.

FIG. 18 illustrates a side view of a well with a temperature controlelement and temperature measurement element in thermal contact with theinterior of the well.

FIG. 19 illustrates an alternative embodiment for sensing the dissolvedoxygen and/or pH level in the wells.

FIG. 20 illustrates an embodiment in which a drip valve is used with thewell plate.

DETAILED DESCRIPTION

In accordance with an embodiment of the present invention, a well plateand supporting instrumentation provides performance capabilities oflarger scale fermenters, such as independently controlling the dissolvedoxygen and/or pH level in each well and independently controlling thetemperature in each well. A well plate in accordance with the presentinvention may be used for controlling and measuring cell growth, whichis useful, e.g., for cell culture, fermentation, and cell based assays.

A well plate and supporting instrumentation, in accordance with thepresent invention, delivers better control over the micro-environmentand thus is more suitable for cell culture, fermentation, and cell basedassays than conventional well plates. Improved control over themicro-environment leads to enhanced signal to noise which can generatemore reliable results or increase throughput. The use of a system withbetter control over the micro-environment can also be used downstream ofa conventional high throughput screen to validate and refine positiveresults.

FIGS. 1 and 2 illustrate a top perspective view and a top plan view of awell plate 100 in accordance with one embodiment of the presentinvention. Well plate 100 is illustrated as having a top surface 102 anda plurality of wells 110, e.g., 24, 48, 96 or any other desired numberof wells that extend generally downward from the top surface 102. Ingeneral, the dimensions and form of well plate 100 may be similar to thetype purchased from Corning Costar from Acton, Mass., as part number#3527 or from Nalge Nunc International from Rochester, N.Y., as partnumber 142485. Well plate 100 may be manufactured from, e.g.,polystyrene, and have a length of 128 mm and a width of 86 mm, with awell volume of 3.4 mL. Of course, many different types of configurationsand dimensions may be used with the present invention. By way ofexample, the well plate 100 may have wells 110 that extend generallyupward from a surface rather than extending downward from the topsurface 102 as illustrated in FIG. 1.

It should be understood that while FIGS. 1 and 2 illustrate the wells110 as round, other geometries may be used if desired. For example,wells 110 may be, e.g., square, which advantageously offer more volumeand better mixing due to turbulence from the corners. The bottom surfaceof the wells 110 is generally flat, but a round well bottom may be usedas well, e.g., the well 110 may have a generally semi-spherical shape.Moreover, the depth of the wells may be varied, which providesadditional well volume with a fixed well plate footprint.

FIG. 3A is a view of the interior bottom surface of one well 110. As canbe seen in FIGS. 2 and 3A, each well 110 includes a plurality ofapertures. The apertures in the bottom surface of each well 110 provideaccess for temperature measurement and control and the dissolved oxygenand pH control for each well.

In one embodiment, a plurality of apertures 112 are located in theapproximate center of the each well 110 and are used provide a gas tothe well 110. The gas that is provided through apertures 112 are used tocontrol the dissolved oxygen and pH level of the contents of the well.The gas is supplied through apertures 112 through a membrane that may belocated on the exterior bottom surface of the well 110. In someembodiments, the membrane may be located on the interior bottom surfaceof the well 110. The membrane and gas supply in general will bediscussed in more detail below. The central apertures 112 areillustrated as rectangular with rounded corners, however, otherdimensions may be used. The central apertures 112 may be considered asingle large aperture with a series of support ribs, whichadvantageously limit the deformation of the external membrane during thegas exchange.

FIG. 3B is a view of another embodiment of the interior bottom surfaceof a well 110. As can seen in FIG. 3B, the gas supply apertures 112 a isan array of circular apertures, e.g., that are 0.2 to 1.0 mm in diameterand spaced approximately 1 mm to 2 mm apart. The use of an array ofapertures 112 a is particularly useful to control the number of bubblesand bubble size during the supply of gas when a porous membrane is used.

Each well also includes two additional apertures 114 and 116, which areused to provide thermal contact between the interior of the well 110 anda heater element and temperature measurement element through themembrane. As illustrated in FIG. 3A, the apertures 114 and 116 arelocated on opposite sides of the well 110 to maximize their distance andto minimize local localized heating errors. Heating and temperaturemeasurements of the contents of a well 110 will be discussed in moredetail below.

The apertures 112, 114, and 116 may be formed in the bottom of the wellplate 100 using conventional cutting techniques such as water jetcutting or laser, which is particularly useful when the well plate 100is manufactured from a plastic. If desired, other cutting techniques maybe used, such as conventional machining. Further, if desired, the wellplate 100 may be custom molded, which obviates the need for cutting theapertures in the bottom of the wells 110.

In addition, each well 110 includes one or more sensors to measure thedissolved oxygen and/or the pH level. The measurement of dissolvedoxygen and/or pH level in each well 110 may be used to control thecontrol the supply of gas to the well 110, e.g., in a feedback loop. Inone embodiment, the sensors are integrated into the well plate 100 asillustrated in FIGS. 3A and 3B. By way of example, an oxygen sensor 140and a pH sensor 142 are deposited on the interior bottom surface of thewell 110. The sensors 140 and 142 may be, e.g., fluorescent tags. Thesensors can be deposited as small dots that are approximately 50 μmthick and 2 mm in diameter. The use of fluorescent tags as integratedsensors in well plate 100 is advantageous as they are inexpensive, anddo not require significant calibration.

The chemicals used to produce the fluorescent tag for oxygen sensor 140may be purchased from, e.g., Aldrich or Precision Sensing GmbH, locatedin Germany. The dissolved oxygen sensor may be based, e.g., on anorganic indicator, or if desired based on Tris(4,7-diphenyl-1,10-phenanthrolin)ruthenium(II).

The chemicals used to produce the fluorescent tag for pH sensor 142 maybe purchased from Precision Sensing GmbH. An adequate pH sensor andmeasuring technique is described in U.S. Pat. No. 6,602,716, which isincorporated herein by reference. In an alternative embodiment, a dyesuch as a pH sensor dye that is embedded into a film may be depositedwithin the well 110. By way of example, a die that is embedded into afilm may be purchased from Precision Sensing GmbH or Molecular Probes,Inc. of Eugene Oreg.

If desired, additional sensors may be used with well 110. Thus, eachwell 110 may include more than two sensors 140 and 142. By way ofexample, fluorescent sensors that measure CO₂ sensors and glucose may beincluded within the wells 110.

FIG. 4 illustrates a perspective view of the bottom of the well plate110 with a membrane 130 attached to the exterior bottom surfaces of thewells 110 (the bottom of the well 110, with its apertures and thedissolved oxygen and pH sensors on the interior bottom surface of thewells are shown with broken lines). The membrane 130 is a highlypermeable thin membrane through which gasses can be easily passed. Themembrane 130 may be precut to fit over all the wells 110 in one piece,or multiple pieces of membrane may be used to cover one or more wells.The membrane 130 is attached to the well plate 100, e.g., with a siliconpressure adhesive or other appropriate adhesive. Alternatively, themembrane 130 may be attached by ultrasonic or thermal bonding, such asthat produced by Toman Tool Corporation. In one embodiment, the membrane130 includes openings that are associated with the sensors 140 and 142of each well 110 so as to minimize interference.

By way of example, the membrane 130 may be manufactured from silicone orsiloxane polymer. Alternatively, the membrane 130 may be made from ablend of siloxane and a thermoplastic such as polycarbonate to increaserobustness. The use of the rectangular central apertures 112,illustrated in FIG. 3A, is particularly useful with a silicon membrane.Adequate silicone or siloxane polymer membranes may be purchased, e.g.,from Specialty Silicone Products, located in Ballston Spa, N.Y. Theparticular membrane 130 will depend on the desired permeability. By wayof example, a membrane that is 50 μm thick may be used, which has apermeability that is approximately thirty times better than carbon basedpolymers. Such a 50 μm thick membrane would provide, e.g., 4×10⁻⁴ mol ofoxygen per hour in each well, where the exposure area is 5 cm² and a 10psi gas pressure is used. With the presence of water on one side of themembrane 130, e.g., in the well 110, the supply of oxygen may bereduced, but will still be several times higher than necessary for avigorous fermentation in the approximately 1 ml of volume in each well110. The use of a permeable membrane is advantageous for gas transfer asthe oxygen goes directly into solution and, thus, no bubbles are formedand lost through the top of the well. Thus, a permeable membrane isuseful for low flow experiments over an extended time period.

Where reactions require higher gas fluxes, a porous membrane 130 may beused, e.g., a membrane that includes small holes. The use of the arrayof circular apertures 112 a, illustrated in FIG. 3B, is particularlyuseful with a porous membrane. By way of example, a membrane thatincludes holes less than 0.2 μm may be used. Liquid cannot pass throughholes of this size nor can microbes that could contaminate thefermentation. In general sterile filtration calls for a pore size ofless than 0.2 μm. Manufacturers of useful membranes include W.L. Gore &Associates, located in Newark Del., Porex Corporation located inFairburn, Ga., and Mitsui Chemicals, Inc. located in Japan. By way ofexample, a 0.2 μm (or smaller) pore size membrane from a material calledePTFE that is laminated with a polyester support and manufactured byW.L. Gore & Associates may be used. The polyester support is useful toprovide strength and it is able to withstand gamma irradiation, which isused to sterilize the well plate 100. Porous membranes are particularlyadvantageous where a high gas transfer rate is desired. Moreover,because of the high gas transfer rate, bubbling will occur which isuseful in stifling the reactor volume. However, because bubbling mayresult in splashing and foam generation, an anti-foaming agent may needto be added to the fermentation.

In one embodiment of the present invention, a laminate of two membranesor a one membrane and another material, such as polyester, may be usedat the same time. FIG. 5A illustrates a side view of two wells 110 witha first membrane 130 and a second material 132, which may be anothermembrane, attached to the exterior bottom surface of the wells 110. Inone embodiment, the first membrane 130 is a porous membrane that isrelatively thick and thus may be used as support for the second membrane132. The second membrane 132 may be, e.g., a thin coating that isapplied to the first membrane 130 to seal the membrane. The secondmembrane 132 may be applied locally at the bottom of each well 110 orover the entire surface of the first membrane 130. The use of a secondmembrane 132 or a coating on the first membrane 130, produces astructure that behaves like a non-porous membrane but that can achieve ahigh gas transfer rate thane with thicker polymer membranes.

It should be understood that the membrane may have alternativeconfigurations. For example, instead of a large sheet of membrane thatcovers the entire bottom of the well plate 110 as illustrated in FIG. 4,the membrane may be formed from individual disks, where each disk coversthe exterior bottom surface of an individual well 110. Alternatively,the membrane may be individual disks that are inserted into individualthe well to cover the interior bottom surface of the well. FIG. 5Billustrates a side view of two wells 110 with membranes 130 a attachedto the interior bottom surface of the wells 110. The membranes 130 a maybe similar to the membranes discussed above and may be ultrasonically orthermally bonded to the wells 110. If desired other bonding techniquesmay alternatively be used, such as a silicon pressure adhesive.Alternatively, the membrane may be formed by individual disks that arepartially embedded into the bottom surface of the well 110. For example,the bottom surface of the well 110 may include a counter bore into whichan individual membrane is mounted. The use of a counter bore in the wellwould countersink the membrane.

Additionally, multiple membranes may be used for different portions ofan individual well 110. For example, a thin highly porous membrane maybe used to cover the apertures 112 for the gas supply to the well, whilea thicker, more robust membrane, e.g., that is optimized for thermaltransfer, may be used to cover apertures 114 and 116 for heater elementand temperature measurement element. Alternatively, a single membranehaving different thicknesses may be used. By way of example, a portionof a silicone membrane that covers the gas supply apertures 112 may berelative thin while the portion of the same membrane that coversapertures 114 and 116 (which are used to provide thermal contact betweenthe interior of the well 110 and a heater element and temperaturemeasurement element) may be relatively thick.

Once the well plate 100 is formed, including the formation of theapertures and the sensors in the well 110, the well plate 100 issterilized, e.g., by exposing the well plate to gamma radiation orethylene oxide.

FIG. 6 shows a block diagram of a device 150 that may be used with wellplate 100 to controls the culture and/or fermentation of the contents ofthe wells. The device 150 includes a compartment 152 in which the wellplate 100 is inserted, e.g., through a top door 154 or through a sidedoor 156. A robotic arm (not shown) may be used to assist in placing thewell plate 100 in the compartment 152. The well plate 100 is clampeddown on an instrumentation block 200, which will be discussed in moredetail below. The environment within the compartment 152 is controlled,e.g., using a gas vent, a TEC based heater/cooler, and a humidifier. Inaddition, the device 150 may include an agitator 158 that moves the wellplate 100 and instrumentation block 200 in, e.g., an orbital pattern.The compartment 152 is separated from an electronics compartment 160 bya bellows, e.g., between the instrumentation block 200 and the walls ofthe device 150, which permits the well plate and instrumentation block200 to move and contains the desired environment within the compartment152, thereby avoiding condensation and other problems with sensitiveoptics and electronics contained within the electronics region 160. Ingeneral, controlling the environment within a chamber, such ascompartment 152 and providing agitation to an element within thechamber, is well within the abilities of those skilled in the art. Thedevice 150 may include a user interface (not shown) that is, e.g.,coupled to the control system for the device and permits the user toprovide input and provides feedback to the user.

FIG. 7 illustrates a side view of one embodiment of vacuum clamping thewell plate 100 to a top surface of the instrumentation block 200. Anumber of pipes 172, e.g. four, extend from a vacuum pump 174 to a flatsurface of the well plate 100. The pipes 172 may include rubber gasketsat the top surface to provide a seal with the well plate 100. When thevacuum is applied to the well plate 100, the bottom surfaces of thewells 110 are placed in firm contact a top surface 180 of theinstrumentation block 200. It should be understood that the pipes 172extend through the membrane 140, which is not shown in FIG. 7. Ofcourse, other methods of clamping the well plate 100 to theinstrumentation block 200 may be used if desired, such as by mechanicalclamping.

FIGS. 8 and 9 illustrate a perspective view and side view, respectively,of a well plate 100 mounted on the instrumentation block 200. The topsurface of the instrumentation block 200 to which the well plate 100 isclamped is a support plate 201. The support plate 201 creates a sealwith the well plate 100 and also provides the temperature controlelements, i.e., a heating/cooling element and a temperature measurementelement. An optical plate 250 is positioned below the support plate 201and includes optical devices that are used to measure the dissolvedoxygen and/or pH level using the sensors 140 and 142. Below the opticalplate 250 is a gas manifold 300, which is illustrated schematically inFIGS. 8 and 9. Manifold 300 is used to control the flow of gas to theindividual wells 110 in the well plate 100.

FIG. 10 illustrates a top perspective view of a support plate 201. Inuse, the support plate 201 is robustly mounted to the gas manifold 300with mount hardware such as bolts. The gas manifold then pulls the wellplate 100 down onto the support plate 201 by vacuum or other comparableclamping methods, as discussed above. As can be seen in FIG. 10, supportplate 201 includes a plurality of similarly configured areas, each ofwhich is associated with a separate well 110 of the well plate 100. Forthe sake of clarity, the footprint of the bottom of a well 110 when thesupport plate 201 is clamped to the well plate 100 is illustrated with adotted line 203 in FIG. 10.

The support plate 201 is, e.g., a printed circuit board 202 that has agasket 204 mounted on the top surface, e.g., with adhesive. The gasket204 provides a gas-tight seal with the well plate 100 when the supportplate 201 is clamped into contact with the bottom of the well plate 100through the membrane 130. The support plate 201 also includes aplurality of heating elements 206 and temperature measurement elements208 on the top surface, where a pair of heating elements 206 andtemperature measurement elements 208 is associated with each well 110.Thus, for example, where the well plate 100 has 24 wells 110, thesupport plate 201 includes 24 pairs of heating elements 206 andtemperature measurement elements 208. As illustrated in FIG. 10, thegasket 204 is trimmed to expose the heating elements 206 and temperaturemeasurement elements 208, which are mounted on the printed circuit board202. By way of example, the gasket 204 may includes cut out sections 204a to expose the underlying heating elements 206 and temperaturemeasurement elements 208. Additionally, the gasket 204 may not extend tothe sides of the board 202, again to ensure that the heating elements206 and temperature measurement elements 208 are not covered.

The heating elements 206 may be, e.g., resistive heater elements. Wherecooling of the contents of the well is desired, the heater elements 206may be Peltier coolers (TEC cooler) instead of resistive heaterelements. Thus, the elements 206 may be used to heat or cool thecontents of the wells 110, the elements 206 will sometimes be referredto as temperature control elements. The temperature measurement elements208 may be, e.g., thermistors.

It should be understood that while the device 150 (shown in FIG. 6)controls the temperature of the overall compartment 152, the heatingelements 206 and temperature measurement elements 208 are used toindependently control the temperature within the individual wells 110.

As can be seen in FIG. 10, the support plate 201 also includes a centralaperture 210, and two side apertures 212 and 214 through both theprinted circuit board 202 and the gasket 204. When properly positioned,the central apertures 210 are aligned with the center apertures 112 ineach well 110. The side apertures 212 and 214 provide optical access tothe sensors 140 and 142 from the optics plate 250, which is positionedbelow the support plate 201. Thus, when properly positioned, apertures212 and 214 are positioned under the oxygen sensor 140 and a pH sensor142 in each well 110.

FIG. 11 is a top plan view of the optics plate 250. Optics plate 250includes a plurality of detection heads 251 that are used in conjunctionwith the sensors 140 and 142 to measure the dissolved oxygen and pHlevel of the contents in a well. By way of example, each detection headincludes a light emitting diode (LED) 252 and a photodiode 254, and aseparate detection head is provided for each sensor in the well plate100. By way of example, the detection heads 251 may use and a Nichiablue-green LED (NSCE310T) (505 nm) for the oxygen sensor 140, a NichiaBlue LED (NSCB310T) (470 nm) for the pH sensor 142, and a HamamatsuS8729-10 photodiodes. A wavelength filter 256 may be mounted over eachphotodiode 254. By way of example, the detection heads 152 may use acolor glass long pass filter that is 1 mm thick and for the oxygensensor 140 may be, e.g., RG630, which passes light of wavelengths longerthan 630 nm and for the pH sensor 142 may be, e.g., OG530, which passeslight of wavelengths longer than 530 nm.

When properly positioned, the LED 252 and photodiode 254 are alignedwith the aperture 212 or 214 and the corresponding sensor 140 or 142 inthe well 110. In addition, optics plate 250 includes an aperture 258that is aligned with central apertures 210 of the support plate 201.

To measure the dissolved oxygen and pH level in the contents of a well,the decay lifetime of the fluorescent oxygen sensor 140 and a pH sensor142 in the well 110 is measured, which corresponds to the amount ofdissolved oxygen and pH level. Measurement of the time response of thesensors 140 and 142 is performed by, e.g., pulsing light from the LED252. For example, the decay lifetime can be measured to determine thedissolved oxygen content. The pH level can be determined by measuringthe intensity ratio between a short lifetime pH indicator and a longlifetime reference indicator.

The pulsed light may be, e.g., a square wave on-off measurement profilemay be used with approximately 1 kHz for the oxygen sensor 140 andapproximately 8 kHz for the pH sensor 142. It is desirable for theperiod of the square wave to be much greater than the lifetime of decaylifetime being measured. Models indicate that the use of a square wavewith a period that is approximately 20 times greater than the decaylifetime being measured provides an error of approximately 1% or less.The square wave is generated using digital techniques, such as using anoscillator circuit with a divider circuit to divide down theoscillations to the desired frequency. Alternatively, a sine wave may beused. The use of a square wave, however, provides a stable frequency,which is relatively difficult to do with a sine wave. Moreover, becausea square wave is “on” and “off”, the use of a square wave advantageouslyavoids problems associated with LED non-linearities.

In general, the fluorescent sensors absorb the incident light and emitlight with a different wavelength after a delay that corresponds to thedecay lifetime. The light emitted from the sensors is then detected bythe photodiode 254 and the phase shift between the incident light andthe emitted light can then be measured. The filter 256 ensures that thephotodiode 254 receives only light emitted from the associated sensorand not light from the LED 252. If desired, instead of measuring thedecay lifetime, the intensity of the light emitted from the fluorescingsensors may be measured. However, measuring the intensity of theresulting light requires calibration to account for thickness, densityand efficiency variations.

The response in the photodiode 254 is measured using a conventional“lock-in” detector, which is well known in the art. Lock-in detection iscommonly performed with an I-Q demodulator circuit, in which two signalsare generated. One signal is the in-phase (I) signal and the othersignal is the quadrature (Q) signal. The amplitude of the signal that isin phase (I) relative to a reference signal is measured along with theamplitude of the signal that is 90 degrees out of phase, i.e., thequadrature (Q) signal, relative to the reference signal. The I and Qmeasurements can then be used to determine, e.g., the amplitude and thephase shift, e.g., using analog electronics or simply by digitizing andprocessing the signal at a high rate (e.g., 10 MHz) in a computer ordigital signal processor.

With the use of a square wave signal, where the lifetime being measuredis much shorter than the period of the square wave, the dissolved oxygencontent can be determined by the following:

$\begin{matrix}{{Amplitude} = {2*\left( {I + Q} \right)}} & {{eq}.\mspace{14mu} 1} \\{{{Lifetime}(\tau)} = \frac{T}{4\left( {\frac{I}{Q} + 1} \right)}} & {{eq}.\mspace{14mu} 2}\end{matrix}$

where I is the in phase signal, Q is the quadrature signal, and T is theperiod of the square wave. Using the lifetime (τ) for the dissolvedoxygen signal, the dissolved oxygen or pH level can be determined usingthe Stern Volmer equation, which is expressed as follows:

$\begin{matrix}{\frac{\tau_{0}}{\tau} = {1 + {K_{SV}\left\lbrack O_{2} \right\rbrack}}} & {{eq}.\mspace{14mu} 3}\end{matrix}$

where τ_(o) are the intrinsic lifetime (no oxygen quenching) fluorescentlifetime for the particular sensor fluorophore and K_(sv) describes asimple linear relationship with the quenching and the oxygenconcentration. If desired, modified Stern Volmer equations may be used,which are well known in the art.

The pH sensor 142 includes an indicator material and a referencematerial, which have different lifetimes for decay. The lifetime ofdecay for the indicator material is a function of pH level, while thelifetime of decay of the reference material does not vary. With the useof a square wave, the pH level can be determined by the following:

$\begin{matrix}{{{Ratio}\mspace{14mu} {of}\mspace{14mu} {indicator}\mspace{14mu} {to}\mspace{14mu} {reference}} = {{\frac{4\tau_{ref}}{T}\left( {\frac{I}{Q} + 1} \right)} - 1}} & {{eq}.\mspace{14mu} 4}\end{matrix}$

where τ_(ref) is the lifetime of the decay for the reference material. Adescription of the pH material and the use of the ratio of indicator toreference to determine pH level can be found in U.S. Pat. No. 6,602,716,which is incorporated herein by reference.

FIG. 12 illustrates a side view of a well 110 with a sensor 140, aportion of the support plate 201 and a portion of the optics plate 250with a detection head 251 including an LED 252 and photodiode 254. Ascan be seen, the LED 252 may be offset from the sensor 140 slightly. Thelight emitted by LED 252, illustrated by cone 253 is incident on sensor140 after passing through aperture 212 in the support plate 201. Lightthat is emitted by the sensor 140 is received by photodiode 254 afterpassing through filter 256. The detection head used with the pH sensor142 may be similarly positioned. Alternatively, the LED 252 may beangled on the optics plate 250 so that the emission is centered on thesensor 140.

It should be understood that other embodiments of the detection heads251 may be used. By way of example, FIG. 13A illustrates an embodiment,in which a lens 260 is used to focus the light on the sensor 140. Afilter 258 may be used with the LED 252 if desired, e.g., if theemission profile of the LED is too broad. By way of example, a shortpass or band pass filter 258 may be used. FIG. 13B illustrates anotherembodiment, in which a beam splitter 262 is used with the detection head251.

In another embodiment, optical fibers may be used with the detectionheads. FIG. 14 illustrates a top plan view of the use of a plurality ofoptical fibers 272 a, 272 b, 272 c, 272 d, 272 e, and 272 f that extendfrom a light source 274 that includes a multiplexer 276 and LED 278 tolocations under each sensor in the wells. The optical fibers may beplastic optical fibers that are, e.g., 1 mm to 2 mm in diameter. Itshould be understood that for a system with 24 wells with two sensorsper well, FIG. 14 shows only half of the necessary fibers. Opticalfibers 280 a, 280 b, 280 c, and 280 d extend from locations under eachsensor in the wells to a detector 282 that includes a multiplexer 284and a photodiode 286. The detector 282 may also include a filter. Bymultiplexing the light source 274 to the different columns and thedetector 282 to the different rows, each sensor can be individuallyexamined. Of course, if desired, the multiplexers 276 and 284 may beeliminated by using a dedicated LED and/or photodiode onto each fiber.

In another embodiment, a single detection head 290 may be used with atwo dimensional stage 292 as illustrated in FIG. 15. Optical fibers 294and 296, which are coupled to an LED 295 and photodiode 297 respectivelyare mounted on the two dimensional stage 292. The stage 292 moves thefibers as illustrated in FIG. 15 to separately examine each sensor inthe well plate 100.

Referring back to FIGS. 8 and 9, gas is supplied to the wells 110 of thewell plate 100 through a manifold 300. The manifold includes gas inputs302 a, 302 b, and 302 c (collectively 302), through which the desiredgas is supplied to the manifold 300. As illustrated in FIG. 9, withinthe manifold 300, separate gas lines 304 a, 304 b, 304 c (collectively304) are routed from the gas inputs 302 to valves 306 a, 306 b, and 306c (collectively 306). It should be understood that, although FIG. 9illustrates the lines overlapping in sections, the gas lines 304 a, 304b, and 304 c are all separate lines. The valves 306 are operated bysolenoids 308 a, 308 b, and 308 c (collectively 308), which may bepurchased from Bio-Chem Valve, Inc., located in Boonton, N.J., orPneutronics division of Parker. Alternatively, micro-electro-mechanicalsystems (MEMs) based valves may be used, such as that manufactured byRedwood Microsystems.

The manifold 300 may include an internal or external filtration andregulation system. By way of example, the manifold may include a filterto filter the incoming gas and a regulator to regulate the gas supply,e.g., to between 5-20 psi. By way of example, the gas supply may beregulated to 5 psi when a porous membrane is used and to a higher psi,e.g., 20 psi, when a silicone membrane is used. The manifold 300 mayalso include a flow limit valve on the gas input lines to limit themaximum flow rate of the gas, e.g., to between 0.01 sccm to 1.0 sccm. Inaddition, a check valve may be included to prevent contamination of thegas supply from back flow from the wells if a membrane were tomalfunction. In one embodiment, the regulator and flow limit valve areadjustable, e.g., through a computer interface or mechanically, so thatwell plates 100 with different types of membranes may be used with thedevice 150.

From the valves 306, a single gas line 310 extends to the top surface312 of the manifold 300, through the central aperture 258 in the opticsplate 250, through the center aperture 210 in the support plate 201 andto the bottom of a well 110. The gas that is provided through the gasline 310 passes through the membrane 130 and into the well 110 throughapertures 112 in the bottom of the well 110.

It should be understood that FIG. 9 illustrates only one set of gaslines for a well 110. The manifold 300 includes separate gas lines304/310, valves 306, and solenoids 308 for each well 110 in the wellplate 100 so that the supply of gas to each well can be independentlycontrolled.

By way of example, one gas line 302 a may supply oxygen which will alterthe dissolved oxygen in the contents in a well 110. The oxygen may besupplied as pure oxygen or as compressed air. Compressed air isadvantageous because it is inexpensive and non-flammable. However,compressed air includes only 20% oxygen and thus, a greater volume ofgas must be provided to the well 110 in order to provide the desiredamount of oxygen.

Another gas line 302 b my supply CO₂ which is used to control the pHlevel of the contents in a well 110. The CO₂ will drive the solutionacidic as it forms carbonic acid in an aqueous solution. Another gasline 302 c may supply NH₃, which is also used to control the pH level ofthe contents in a well 110. The NH₃ will drive the solution basic. TheNH₃ may be supplied, e.g., as either pure ammonia gas or diluted (10:1)with nitrogen, which is commonly done for safety.

Other gases may also be supplied, such as nitrogen or other inert gasesthat can be used to purge the wells of oxygen in a low-oxygenapplication, e.g., by bubbling the inert gas through the contents of thewell, or to provide bubbling action without introducing chemicallyactive elements. Additionally, methane and/or hydrogen may also beprovided. Of course, other gases may be provided if desired.

It should also be understood that while FIGS. 8 and 9 illustrate threevalves 306 per well 110, fewer valves may be used if desired. Forexample, one valve may be used for controlling the oxygen/air supply,while a second valve may be used to control either CO₂ or NH₃. By way ofexample, the oxygen/air supply may be provided through apertures 112 inthe bottom of the well 110, while pH regulation may be accomplishedusing a micro-valve to drip in dilute NaOH or acid.

It should be understood that other methods may also be used to supplygas to the wells 110. For example, chemical reactions, electrolysis, andthermal devolution, in which an element releases gas as it is heated,may be used.

The gas supply and sensors may be linked together in a feedback loop.FIG. 16 schematically illustrates a feedback loop for one well 110. Asillustrated, in FIG. 16, a detection head on the optics plate 250 iscoupled to a processor 390 that is also coupled to a solenoid 308. ThepH level in the contents of the well 110, as measured by the sensor 142and detection head, is determined by the processor 390. The processor390 controls the solenoid 308 to provide the appropriate amount of gasto the well 110 to produce the desired pH level. The detection andcontrol of the dissolved oxygen content is controlled in a similarmanner.

Additionally, the temperature of the contents of the well 110 iscontrolled in a feedback loop. FIG. 17 illustrates top view of a portionof the well plate 100 with a well 110 over the support plate 201. Themembrane 130 is not shown so that the temperature control element 206and temperature measurement element 208 can be seen through theapertures 114 and 116, respectively. As illustrated, the temperaturecontrol element 206 and temperature measurement element 208 are coupledto the processor 390. In order to control the temperature of thecontents of a well, the processor 390 controls the temperature controlelement 206 based on the temperature measurement element 208measurements. The temperature control elements 206 and temperaturemeasurement element 208 for each well are individually coupled to theprocessor 390 so that the temperature in each individual well 110 can beindependently controlled.

It should be understood that the temperature control elements 206 andtemperature measurement element 208 may be in thermal contact with theinterior of the well 110 through a surface wall of the well. By way ofexample, FIG. 18 illustrates a side view of a well 110 a along with aportion of the support plate 201 and temperature control element 206 andtemperature measurement element 208 in thermal contact with the interiorof the well 110 a. The well 110 a is similar to well 110 in FIGS. 2 and3, but does not include apertures that extend through bottom surface forthe temperature control and temperature measurement elements 206, 208.Well 110 a includes indentations 452 and 454 in the exterior of thebottom surface 451. The indentations are at least partially filled witha thermally conductive material 456, such as a conductive siliconmaterial. An adequate thermally conductive material is referred to asGap Pad and may be purchased from Bergquist Co., located in ChanhassenMinn. The temperature control elements 206 and temperature measurementelement 208 are in thermal contact with the interior of the well 110 athrough the thermally conductive material 456 and the bottom surface 451of the well 110 a. In some embodiments, the thermally conductivematerial 456 is not used and the temperature control elements 206 andtemperature measurement element 208 are in thermal contact with theinterior of the well 110 a through the surface 451 of the well 110 a.

FIG. 19 illustrates an alternative embodiment for sensing the dissolvedoxygen and/or pH level in the wells 110. As illustrated in FIG. 19,probes 500 can be physically inserted into the wells 110 in order tosense the dissolved oxygen and/or pH level. Probes 500 may, e.g., extenddownward from the lid 502 of the well plate 100 and are inserted intothe media in the wells 110 when the lid is placed on the well plate 100.The probes 500 are connected to the processor 390 when the well plate100 and lid 502 are placed in the device 150.

In one embodiment, probes 500 can be used for oxygen sensing. By way ofexample, an oxygen sensing probe may be a polarographic (Ross or ClarkCell) and galvanic cell probes. Manufactures of probes that may be usedfor this purpose include Diamond General and Broadley James. Apolarographic or galvanic probe 500 may be also be used with the wellfrom the bottom, where the electrolytes and electrodes of thepolarographic or galvanic probe 500 is separated from the media in thewell 100 by the membrane 130. In such an embodiment, an additionalaperture in the well 110 would be necessary for each probe.Alternatively, probes 500 may be an optical fiber with a fluorescentmaterial attached to the end of the fiber. The approach is similar tothe sensors 140 and 142 discussed above, but the sensor is attacheddirectly to the fiber. A manufacturer of an optical fiber probe that maybe used is Ocean Optics.

Where the probes 500 are used to sense the pH level in the mediacontained in the wells 110, the probe may be a “glass electrode”. Glasselectrodes are manufactured from a glass that has an electrostaticpotential that is dependent on the environmental pH. Alternatively, theprobe 500 may include an ion-sensitive FET (ISFET), which is sensitiveto the environmental pH. A manufacturer of an ISFET that may be used isSentron. In another embodiment, the probe 500 may be a transmissionprobe that uses a pH dye on an embedded film. Light is passed throughthe film with the dye and the transmission is measured, which indicatesthe pH level. Ocean Optics manufactures transmission probes that may beused. The light source for the transmission probe may be either an LEDor a white light source, such as a flash lamp. A photodiode is used todetect the light. The measurement may be made at two wavelengths. Theratio of the two wavelengths provides information as to the pH level. Inorder to perform two measurements at different wavelengths, either twolight sources, two detectors (each with a filter), or a spectrometer isneeded.

If desired, different types of sensors may be used to measure thedissolved oxygen and pH level. Thus, for example, the dissolved oxygenmay be measured using sensor 140 while the pH is measured with probe500. Alternatively, both the dissolved oxygen and pH level may bemeasured using probes.

Additional measurement devices may be used with the present invention.For example, it may be desirable to measure cell density, e.g., usingoptical density and/or impedance. Further, it may be desirable tomeasure the concentration of fluorescently tagged protein or substrateduring fermentation.

In another embodiment, the pH level is controlled using a liquid dripvalve instead of a gas supply. FIG. 20 illustrates a side view of a wellplate 550, which is similar to well plate 100, except that the wells 560do not include a gas supply aperture in the bottom surface. Asillustrated in FIG. 20, a series of drip valves 570 are positionedrelative to the well plate 550 such that at least one drive valve 570 isheld over the wells 560. The drip valves 570 may extend and/or may beheld by the lid 552 of the well plate 550. The drip valves 570 arecoupled to a supply 572, which provides the desired liquid to the dripvalves 570 to adjust the pH level in the contents of the well 560, suchas dilute NaOH or acid. The drip valves are coupled to and controlled bythe processor 390. The detection heads 251 associated with each well 560provide information to the processor 390 regarding the pH level ofindividual wells. In response the processor 390 controls the flow ofliquid into the wells 570 to adjust the pH level to the desired level.The drip valves 570 may be, e.g., peristaltic or syringe pumps or amicro valve. If control over the dissolved oxygen is desired, a gassupply may be provided to the well, e.g., through an aperture in thebottom of the well, as described above.

Although the present invention is illustrated in connection withspecific embodiments for instructional purposes, the present inventionis not limited thereto. Various adaptations and modifications may bemade without departing from the scope of the invention. Therefore, thespirit and scope of the appended claims should not be limited to theforegoing description.

1. An apparatus used with a well plate that has a plurality of wells,each well being defined by at least one surface that defines an interiorcavity having an opening, the apparatus comprising: a plurality of dripvalves, wherein there is at least one drip valve associated with eachwell positioned over the opening of each well, the drip valvesconfigured to provide a liquid to the interior cavities of theassociated wells; a plurality of detectors for detecting a property ofthe contents of the wells, wherein there is at least one detectorassociated with each well; and a control system coupled to the pluralityof detectors and the plurality of drip valves, the control systemcontrolling the amount of the liquid provided by the drip valves to theassociated wells in response to the property of the contents in theassociated wells detected by the detectors associated with each well. 2.The apparatus of claim 1, wherein the liquid is one of NaOH and acid. 3.The apparatus of claim 1, further comprising a gas supply for providinggas to the interior cavities of the associated wells, wherein thecontrol system is coupled to the gas supply, the control systemcontrolling the amount of gas provided by the gas supply to theassociated wells in response to the property of the contents in theassociated wells detected by the detectors associated with each well. 4.The apparatus of claim 1, wherein each well in the well plate includesat least one fluorescent material disposed within the well, the at leastone fluorescent material reacts to the property of the contents of thewell, wherein the plurality of detectors comprise: at least one lightsource configured to illuminate the fluorescent material; and at leastone photodetector for detecting the light emitted by the fluorescentmaterial.
 5. The apparatus of claim 1, wherein the light sourceilluminates the fluorescent material at a frequency and wherein detectormeasures the phase delay of the light emitted by the fluorescentmaterial.
 6. The apparatus of claim 1, wherein the plurality ofdetectors comprises at least one probe that extends into the interiorcavity of the well.